U.S. patent application number 10/818585 was filed with the patent office on 2005-10-13 for slit confocal microscope and method.
This patent application is currently assigned to FUJIFILM ELECTRONIC IMAGING LTD.. Invention is credited to Gouch, Martin Philip.
Application Number | 20050225849 10/818585 |
Document ID | / |
Family ID | 34940562 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050225849 |
Kind Code |
A1 |
Gouch, Martin Philip |
October 13, 2005 |
Slit confocal microscope and method
Abstract
A slit confocal microscope comprises a linear light source; a
detection system; and a focusing system for focusing light from the
source onto a sample and focusing returning light onto the
detection system. The detection system comprises a one-dimensional,
linear array of detectors onto which light from the sample is
focused directly or indirectly by the focusing system. A control
system causes the linear array of detectors to integrate light from
a corresponding line of pixels in the sample.
Inventors: |
Gouch, Martin Philip;
(Herts, GB) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
FUJIFILM ELECTRONIC IMAGING
LTD.
Herts
GB
|
Family ID: |
34940562 |
Appl. No.: |
10/818585 |
Filed: |
April 5, 2004 |
Current U.S.
Class: |
359/385 |
Current CPC
Class: |
G02B 21/0024
20130101 |
Class at
Publication: |
359/385 |
International
Class: |
G02B 021/06 |
Claims
1. A slit confocal microscope comprising a linear light source; a
detection system; and a focusing system for focusing light from the
source onto a sample and focusing returning light onto the
detection system, wherein the detection system comprises a
one-dimensional, linear array of detectors onto which light from
the sample is focused directly or indirectly by the focusing
system, and a control system for causing the linear array of
detectors to integrate light from a corresponding line of pixels in
the sample.
2. A microscope according to claim 1, further comprising a mask
defining a slit at the focus of light from the sample.
3. A microscope according to claim 2, further comprising a further
focusing system for focusing the slit onto the linear array of
detectors.
4. A microscope according to claim 1, wherein the linear array of
detectors is located at the focus of the focusing system.
5. A microscope according to claim 1, further comprising means for
moving the array of detectors relative to a sample in an image
plane of the focusing system.
6. A microscope according to claim 1, wherein the detection system
comprises more than one linear array of detectors, the arrays being
substantially parallel with one another, each array of detectors
being sensitive to a respectively different colour.
7. A microscope according to claim 6, wherein the light source is
adapted to generate more than one beam of respectively different
colours, the focusing system focusing returning light to
respectively different focal locations, and wherein the detecting
system is movable to bring each array to the corresponding focal
location of the focussing system.
8. A microscope according to claim 1, wherein the detection system
comprises a single linear array of detectors, and the linear light
source is adapted to generate lines of different colours
alternately, the detection system being sensitive to all colours
generated by the linear light source.
9. A slit confocal microscope according to claim 6, wherein the
colours comprise red, green and blue.
10. A microscope according to claim 1, wherein the linear array of
detectors comprises a CCD array.
11. A method of operating a slit confocal microscope comprising a
linear light source; a detection system; and a focusing system for
focusing light from the source onto a sample and focusing returning
light onto the detection system, wherein the detection system
comprises a one-dimensional, linear array of detectors onto which
light from the sample is focused directly or indirectly by the
focusing system, and a control system for causing the linear array
of detectors to integrate light from a corresponding line of pixels
in the sample, the method comprising: a) illuminating a line of
pixels from the sample, light from the sample being focused onto
the linear array of detectors; b) integrating the incident light on
the detectors corresponding to the line of pixels; and, c)
repeating steps a) and b) with the same linear array for successive
lines of pixels on the sample.
12. A method according to claim 11, further comprising causing
relative movement between all or part of the microscope and the
sample to illuminate different lines of pixels on the sample.
13. A method according to claim 11, the method comprising carrying
out steps a)-c) for one colour and then repeating steps a)-c) for
the same line of pixels but when illuminated with one or more
further colours.
14. A method according to claim 11, the method comprising, for each
line of pixels, carrying out steps a) and b) for each of two or
more colours before carrying out step c).
15. A method according to claim 11 comprising causing continuous
relative movement between the sample and the detection system such
that successive lines of pixels on the sample are focused on to the
detection system; repeatedly illuminating the sample with different
colours at a rate such that a line of pixels illuminated with one
colour overlaps a line of pixels illuminated with the next colour;
and, for each illuminated line of pixels integrating the incident
light on the detectors.
16. A microscope according to claim 2, further comprising means for
moving the slit relative to a sample in an image plane of the
focusing system.
17. A microscope according to claim 2, further comprising means for
moving the array of detectors and the slit relative to a sample in
an image plane of the focusing system.
18. A slit confocal microscope according to claim 7, wherein the
colours comprise red, green and blue.
19. A slit confocal microscope according to claim 8, wherein the
colours comprise red, green and blue.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a slit confocal microscope and
method of operating such a microscope.
DESCRIPTION OF THE PRIOR ART
[0002] Confocal microscopes were first described in U.S. Pat. No.
3,013,467 and are now well known in the art. A typical confocal
microscope comprises a light source; a detection system; and a
focussing system for focussing light from the source onto a sample
and focussing returning light onto the detection system. In a
typical example, an object is illuminated from a small area the
size of a single pixel which is confocal with the detector pixel.
FIGS. 1A and 1B illustrate such a conventional arrangement.
[0003] Thus, a point source 1 generates a light beam which impinges
upon a beam splitter 2 from which it is reflected onto a focussing
lens 3. The lens 3 focuses the light onto an object 4 and then
light reflected or emitted by the object passes back to the lens 3
where it is focussed through the beam splitter 2 onto a detector 5
such as a photomultiplier tube located behind a pinhole (not shown)
acting as a field stop. The illumination source 1 is the same size
as the pinhole.
[0004] The system is circularly symmetric so that FIG. 1
illustrates the appearance of the system from the side and in
plan.
[0005] The confocality causes the image of the object to drop in
intensity as the object moves away from the focus. In a
conventional multiphoton system the effect of defocusing is simply
to blur the image. In a confocal system, not only does it blur the
image but also it darkens the image. This means that out of focus
objects do not affect in focus objects.
[0006] An out-of-focus situation is shown in FIG. 2. To produce a
full two-dimensional image, the detector 5 and illumination area is
normally moved across the object 4 in a raster scanning mechanism,
usually with moving mirrors. If three-dimensional images are
required then these can be produced by producing the
two-dimensional images at different focal planes. This
three-dimensional image can then be visualised using a
three-dimensional imaging device or using a three-dimensional
visualisation package. Alternatively, a two-dimensional image can
be produced by merging the two-dimensional images of the different
focus planes and thus produce an image with greater depth of focus
than that which can be produced with a multiphoton microscope with
the same optical resolution.
[0007] Because confocal microscopes are rotationally symmetric
around the optical axis, the light level drop is proportional to
the fourth power of the defocus. This is because the light level
illuminating the object drops proportional to the square of the
defocus and the light level detected from the object drops
proportional to the square of the defocus. A typical rule of thumb
is an image normally continues to look in focus until the PSF
(point spread function) grows to a factor of four over the in focus
condition. This means that in a confocal microscope, the light
level has dropped by a factor of 4.sup.4=256 times by the time the
depth of focus rule of thumb condition has been reached. This
enables confocal microscope images to be added together when
separated by a defocus distance similar to four PSF's and for each
plane to be unaffected by any other plane.
[0008] A hybrid system of multiphoton imaging and confocal imaging
can be generated if the light source is a line of light confocal
with a slit. In this case, we get a blend of confocal and
multiphoton imaging. It is often used for a visual confocal
microscope and a description can be found in the literature such as
"Handbook of Biological Confocal Microscopy", 2.sup.nd Edition,
James B Pawley, Plenum Press. Such a microscope is termed a "slit
confocal microscope".
[0009] The generation of a two-dimensional image is then performed
by optically scanning the slit over the sample. For a digital
image, the literature mentions the use of film cameras, or 2D
detectors, "Handbook of Biological Confocal Microscopy", 2.sup.nd
Edition, James B Pawley, Plenum Press, chapter 12, p195, such as
CCD and cooled CCD for low light level conditions.
[0010] The loss of confocality in one dimension does have an effect
but it is relatively minor. As a first approximation the equation
for light intensity with focus is: 1 I = ( w 0 2 w z 2 ) 2 where (
i ) w z = ( na z > w 0 ) ? na z : w 0 where ( ii ) na = 1.22 2 w
0 where ( iii )
[0011] .lambda.=wavelength of light
[0012] w=pixel size
[0013] z=distance from plane of focus
[0014] na=numerical aperture
[0015] I=intensity
[0016] w.sub.o=pixel size at plane of focus
[0017] w.sub.z=pixel size a distance z from plane of focus
[0018] I.sub.SC=intensity of slit confocal system
[0019] The equations for a semi-confocal system to set first
approximation are
[0020] Using equations (ii) and (iii) 2 I SC = ( w 0 2 R z w z 2 )
2 where ( iv ) R z = - z + sin z where ( v ) z = 2 cos - 1 ( w 0 w
z ) ( vi )
[0021] This gives a fairly similar drop off in intensity with
change in focus as shown in FIG. 3.
[0022] The cross talk between two focal planes is thus increased
for the semi-confocal case but as can be seen in FIG. 4, this is
limited and much less than that for a non-confocal case or a
multiphoton system.
[0023] The use of a 2D detector for a slit scanner has a number of
disadvantages to it. One is that the integration time, the time for
which the detectors accept photons, is set as the time for a full
frame. The longer the integration time the larger the dark noise
from the detector. This is why cooled CCD cameras are used in these
systems as the lowering of the temperature lowers the thermal
noise.
[0024] In accordance with a first aspect of the present invention,
a slit confocal microscope comprises a linear light source; a
detection system; and a focussing system for focussing light from
the source onto a sample and focussing returning light onto the
detection system, wherein the detection system comprises a
one-dimensional, linear array of detectors onto which light from
the sample is focussed directly or indirectly by the focussing
system, and a control system for causing the linear array of
detectors to integrate light from a corresponding line of pixels in
the sample.
[0025] In accordance with a second aspect of the present invention,
a method of operating a slit confocal microscope according to the
first aspect of the invention comprises:
[0026] a) illuminating a line of pixels from the sample, light from
the sample being focused onto the linear array of detectors;
[0027] b) integrating the incident light on the detectors
corresponding to the line of pixels; and,
[0028] c) repeating steps a) and b) with the same linear array of
detectors for successive lines of pixels on the sample.
[0029] In the present invention, in place of the known 2D detector,
we utilize a one-dimensional line scan detector such as a CCD, at
the point of the detector slit, or confocal with it using other
imaging optics, so that the integration time needs only to be as
long as a single line integration time. For example, for a 512 line
image, the noise from the thermal effects is 512 times greater on a
2D detector than that for a line scan detector. If the image is
large, for instance with 10,000 lines then the thermal noise is
10,000 times greater for 2D detectors. There is thus a clear
advantage in using line scan detectors over 2D detectors in that
the noise is considerably lower enabling larger images to be
scanned, or lower light levels to be detected or avoiding the use
of cooling of the detector.
[0030] In order to build up a 2D image, relative movement must be
caused between the line of illumination and the sample and this can
be done in a variety of conventional ways. For example, the sample
can simply be moved relative to the microscope or vice versa.
Alternatively, the linear array of detectors can be moved across
the imaging plane in phase with any slit which is provided in a
moving slit or stationary slit arrangement. Suitable scanning
systems using mirrors or the like are shown in FIGS. 2 and 3 of
"Handbook of Biological Confocal Microscopy", 2.sup.nd Edition,
James B Pawley, Plenum Press, chapter 25.
[0031] A second disadvantage of a 2D detector for a. slit scanner
is when colour imaging is required. Typically 2D CCD cameras are
made from a mosaic of red, green and blue sensitive photo sites.
This means there is no place where a single pixel has all three
colours detected from exactly the same place on the sample. This
leads to a drop in actual resolution of the image. The normal
methods of overcoming this problem in 2D cameras of dithering the
detector does not work well in the slit scanner unless the detector
is dithered between consecutive frame scans of the image which is
slow and difficult as each frame needs to be very accurately
aligned to prevent jitter in the image. Errors of one tenth of a
pixel can be detected and errors of one fifth of a pixel are easily
detected.
[0032] If a line scan detector array is used then colour images can
be produced in two different ways. One is to use a three stripe CCD
detector. This has three lines of detectors which are displaced
from each other by a small number of lines. To do this we need to
illuminate the sample with three lines of different colour e.g.
red, green and blue (RGB) as well so we have in effect three
confocal lines. The lines are then moved across the image and each
colour is then reregistered by delaying or shifting two of the
colours by a suitable amount. This will remove any jitter problems
and as colour registration is much more tolerant to around one
third of a pixel is easier to do and does not require high accuracy
over long periods of time such as a few frames but accuracy over a
short period of time such as a few lines. This also reduces the
sensitivity to the sample moving over the time. In this way, every
pixel on the sample has all three colours detected.
[0033] The other way of using a line scan detector array to produce
colour images is to use a single monochrome line scan detector and
to change the colour of the light from the linear source. For
example, it is possible to change the colour of the light on a line
by line basis in which case the colours will always be one third of
a pixel out of registration which can be compensated for, if
required, by using interpolation methods but does not require any
long term registration to be maintained. Another way of doing this
is to change the colour on a frame by frame basis but this requires
one third of a pixel registration over three frames to produce
acceptable images, which is more difficult.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Some examples of slit confocal microscopes according to the
invention will now be described and contrasted with known
microscopes with reference to the accompanying drawings, in
which:
[0035] FIG. 1 illustrates the primary components of a conventional
confocal microscope;
[0036] FIG. 2 illustrates the microscope of FIG. 1 but with the
object in an out-of-focus position;
[0037] FIG. 3 illustrates for a slit confocal microscope the
variation in intensity from each plane with changing focus for two
planes separated by 100 focus units;
[0038] FIG. 4 illustrates for a slit confocal microscope a confocal
microscope and a multiphoton microscope the intensity of the
changing focus for two planes separated by 100 focus units;
[0039] FIGS. 5A and 5B are a side elevation and plan view of an
example of a slit confocal microscope according to the
invention;
[0040] FIG. 6 illustrates (not to scale) a multi-colour CCD
array;
[0041] FIG. 7 illustrates schematically an alternative illumination
system; and,
[0042] FIG. 8 illustrates part of a multi-colour slit confocal
microscope.
DETAILED DESCRIPTION OF EMBODIMENTS
[0043] FIGS. 5A and 5B illustrate an example of a slit confocal
microscope according to the invention, in side view and plan
respectively. In this case, the source 1. comprises a linear source
10 which generates a line of white light which is focussed by the
lens 3 onto the object where, in plan view, it illuminates a linear
region 11. The returning light is focussed onto a linear detector
array 13 in front of which is provided an optical slit opening 12,
also effectively at the focus of the lens 3.
[0044] The detector 13 is connected to a processor 14 which in turn
is connected to a data store 15.
[0045] In use, the object is illuminated by a line of light from
the source 10 and this causes light reflected from and/or emitted
by the object to be focused onto the linear array of detectors 13,
typically a CCD array. After an integration time, the processor 14
controls the array 13 to transfer the accumulated charge on each
detector to a transfer gate, the charges then being serially
downloaded to the processor 14 and stored in the store 15.
[0046] Although FIG. 5A illustrates the presence of a linear slit
opening 12, this is not essential if the detector 13 is located at
the focal point of the lens 3 and thus the slit opening 12 has been
omitted in FIG. 5B.
[0047] In an alternative arrangement (not shown), the slit opening
12 could be provided with the detector 13 spaced behind the slit
opening and with further focusing optics to focus light received
through the slit opening 12 onto the detector array 13. In other
words, a second confocal arrangement is provided between the
detector 13 and the slit opening 12. This can be useful from a
practical point of view where it is difficult to locate the
detector array 13 at the focal point of the lens 3.
[0048] There are a number of ways in which a two-dimensional image
of the surface of the object 4 can be generated. One approach is to
cause relative bodily movement between the slit confocal microscope
on the one hand and the object 4 on the other with the source 10
illuminating successive lines of pixels on the surface of the
object. Light from those lines of pixels is then stored in a
corresponding array in the store 15.
[0049] In a conventional confocal system, however, this slow scan
is normally generated with the use of a scanning mirror system
which is difficult to make accurately repeatable, uniform in speed
of scanning and to cover a large area. Typically, the image sizes
are 512.times.512 pixels. This approach can be used to advantage in
the invention where the line scanner slit confocal system requires
no moving mirrors in the optical path and is much more repeatable
and easier to make accurate. Also it is easy to scan large areas as
the length of a CCD detector 13 is typically 10000 pixels and the
length of traverse is limited only by how far it is desired to move
the object or the scanning system. Typical scan sizes can be as
large as 10,000.times.10,000 pixels or even longer in the slow scan
direction. Thus, the slit opening 12 or detector 13 is maintained
stationary and a mirror system used to cause light from the light
source 1 to impinge on different lines of pixels on the object
surface 4. Suitable systems are shown in FIGS. 2 and 3 of "Handbook
of Biological Confocal Miscroscopy", 2.sup.nd Edition, James B
Pawley, Plenum Press, chapter 25.
[0050] In another example, a mirror system is provided to cause
successive lines of pixels on the object to be illuminated by the
line of light from the source 1 and the detector 13 is moved across
the confocal plane defined by the lens 3 so as to record light from
each illuminated line of pixels. In other words, the slit in front
of the detector is omitted and the detector array is effectively
moved in phase with the "slit".
[0051] In the examples described so far, the detector has been
monochromatic and the light source 1 has generated a line of white
or monochromatic light. It is possible to modify the apparatus
shown in FIGS. 5A and 5B to handle colour imaging.
[0052] In a first example, the detector 13 is replaced by a
multi-stripe CCD (or other) array 13' shown in FIG. 6. In this
case, three lines or stripes of detectors are provided on a common
support, namely a line of red sensitive detectors 20, green
sensitive detectors 21 and blue sensitive detectors 22. Each line
of detectors is associated with a respective transfer gate
23,24,25. These are individually coupled with the processor 14. In
practice, each line of detectors 20,21,22 is spaced by about 12
such lines from the adjacent line of detectors. In this case, the
line source generates three slit shaped beams of different colours,
typically red, green, blue 40,41,42. These are focused by the lens
3 following reflection by the beamsplitter 2 onto spaced lines on
the object 4. Light from the object 4 is then returned through the
lens 3 and focused through the beamsplitter 2 to three different
confocal locations 43-45 (FIG. 8). Effectively, therefore, three
slit confocal systems are defined.
[0053] The detector array 13' is then traversed from left to right,
as seen in FIG. 8, to bring the appropriate array of detectors in
line with the focused image. Thus, the red sensitive detectors 20
will be aligned with the focus 43 and then the array 13' moved to
bring the green sensitive detectors 21 into alignment with the
focal position 44, and then moved once more to bring the blue
sensitive detectors 22 in line with the focus 45. Following a full
scan, red, green and blue images will be obtained from each line on
the object 4 and these can be rearranged into phase with each other
in a straightforward manner by the processor 14 to generate a
resultant colour for each pixel on the object 4.
[0054] It should be understood that the detector array shown in
FIG. 6 is not being used as a two-dimensional array but as a set of
one-dimensional arrays which obtain light from the same linear
array of pixels on the surface of the object 4.
[0055] An alternative approach is illustrated in FIG. 7 in which a
monochromatic detector array 13 is used but the colour generated by
the light source 1 is varied. This can be achieved in a number of
different ways of which FIG. 7 is one example. In this case, white
light from a linear source 1 passes through one of a set of three
red, green and blue filters 30-32 located on a rotatably mounted
filter wheel 33. In a first stage, the filter wheel 33 is arranged
such that the red filter 30 is aligned with the white light so that
only red light passes through the filter wheel 33 to an
illumination slit 34 prior to impinging on the beam splitter 2.
Information from the object 4 is then recorded by the detector
array 13 and downloaded to the processor 14. The filter wheel 33 is
then rotated to bring the green filter 31 into alignment with the
white light and the process repeated. The process is repeated once
more with the filter 32 in alignment with the white light source.
The three sets of colour information for each pixel in the same
line on the object 4 are then processed as described above.
[0056] In use, consider an example in which the detector 13, lens 3
and illumination slit 34 are all fixed with respect to each other
and the object 4 is moved continuously. If we assume the time to
take a line of red, green and blue pixels is t then each line of
pixels of one particular colour e.g. red when the red filter wheel
is in the illumination path, or green when the green filter in the
filter wheel is in the illumination path, or blue when the blue
filter in the filter wheel is in the illumination path, is captured
in time t/3. The width of the illumination slit 34 is arranged to
be x such that in time t the sample 4 has moved x in relation to
the microscope. Thus we detect a series of red, green and blue
lines x/3 out of phase with each other. This can be corrected if
desired with the use of interpolation.
[0057] Of course, a similar process can be used when other means
are adapted to cause relative movement.
[0058] In another example (not shown), the light source could
comprise a number of differently coloured sources such as LEDs
which are selectively energized to generate light of different
wavelengths.
* * * * *